Cloning of the TgIST gene in T. gondii and subsequent retrovirus transduction were performed at biosafety level 2 with the approval of the Institutional Biosafety Committee of Seoul National University (IBC SNU; approval number SNUIBC-R180523-1).

The murine hepatoma cell line (Hepa-1c1c7) and human fibroblast cell line (HFF) were obtained from the Korean Cell Line Bank (Seoul, Korea). The retrovirus packaging cell line (Phoenix) was purchased from Cell Biolabs (San Diego, CA, USA). Hepa-1c1c7, HFF, and Phoenix cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM; WELGENE Inc., Gyeongsan, Korea) containing 10% fetal bovine serum (FBS; WELGENE) and 1% antibiotic–antimycotic (AA) solution (WELGENE) at 37°C in a 5% CO₂ incubator. To induce an inflammatory environment in hepatocytes, Hepa-1c1c7 vector cells and Hepa-1c1c7 TgIST cells were seeded in a 6-well plate at 3×10⁵ cells/well and incubated for 24 h. The cells were stimulated with 100 ng/ml of IFN-γ/TNF-α for 24 h before conducting the experiment.

Murine IFN-γ and TNF-α were supplied by Peprotech (Rocky Hill, NJ, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan).

The TgIST gene sequence (ToxoDB database, Gene ID: TGRH88_0887790) with BamHI (GGATCC) and EcoRI (GAATTC) restriction sites was amplified through PCR using the cDNA of the T. gondii RH strain. The forward primer sequence was 5′-CCGGATCCCGATGCGTTGGTGTGCC-3′, and the reverse primer sequence was 5′-CCGAATTCCTAGACC CGCCGTTCACTAC-3′. PCR was conducted under the following conditions: 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 3 min, with a final extension step at 72°C for 10 min. The PCR product (2,691 bp) of TgIST was determined via gene sequencing (Cosmogenetech, Seoul, Korea). To create a TgIST-expressing retrovirus plasmid, the amplified TgIST PCR products and pBABE-HAII plasmid (Addgene, Watertown, MA, USA) were digested with restriction enzymes (BamHI and EcoRI) and ligated with T4 DNA Ligase (Sigma-Aldrich, St. Louis, MO, USA). Phoenix retrovirus packaging cells (Cell Biolabs, San Diego, CA, USA) were transfected with either a plasmid vector (pBABE-HAII) alone or pBABE-HAII-TgIST using a Lipofectamine 3000 transfection kit (Life Technologies, Carlsbad, CA, USA) and Opti-Minimal Essential Medium (Life Technologies). Retrovirus-containing supernatants were centrifuged at 2,000 rpm for 15 min at 4°C and filtered through 0.45-μm syringe filters. To generate a Hepa-1c1c7 cell line stably expressing TgIST, pBABE-HAII-Vector or pBABE-HAII-TgIST retrovirus-containing supernatants were treated with 8 μg/ml of polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 48 h. The retrovirus-infected Hepa-1c1c7 cells were then treated with puromycin (2 μg/ml) (Santa Cruz Biotechnology). TgIST expression in Hepa-1c1c7 cells was measured through PCR using the SureCycler 8800 thermal cycler (Agilent Technologies, Santa Clara, CA, USA), and the PCR products were visualized through 1% agarose gel electrophoresis using the Gel Doc XR+ Gel documentation system (Bio-Rad, Hercules, CA, USA).

Stable TgIST expression in Hepa-1c1c7 TgIST cells was confirmed through PCR using TgIST and/or pBABE-HAII-Vector primers, including the primer sequences listed in Supplemenaty Table S1. Total RNA was extracted using the HiGene Total RNA Prep kit (BIOFACT, Daejeon, Korea) and reverse-transcribed into cDNA using the Reverse-Transcription Master Premix kit and oligo d(T)15 primers (ELPIS Biotech, Daejeon, Korea). The cDNA was subjected to PCR to validate TgIST expression using a SureCycler 8800 thermal cycler (Agilent Technologies, Santa Clara, CA, USA), followed by 1% agarose gel electrophoresis with Stay Safe Nucleic Acid Gel Stain (Real Biotec Corporation, Banqiao City, Taiwan).

Hepa-1c1c7 vector cells and Hepa-1c1c7 TgIST cells were seeded in a 96-well plate at 3×10³ cells/well and incubated for 0, 24, 48, 72, and 96 h. The optical density (450 nm, Infinite M200 PRO microplate reader (Tecan)) was measured using the CCK-8 assay kit.

Total RNA was extracted using the HiGene Total RNA Prep Kit (BIOFACT, Daejeon, Korea) and reverse-transcribed to cDNA using the Reverse-Transcription Premix kit (Elpis Biotech, Daejeon, Korea). Real-time qPCR was conducted using the CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories) and TOPreal qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics, Daejeon, Korea). Amplification reactions were performed at 95°C for 15 min, followed by 40 cycles of 95°C for 20 sec, 59°C for 20 sec, and 72°C for 30 sec. SYBR green fluorescent signals were assessed using the iQTM5 optical system software (Bio-Rad Laboratories). The relative expression of target genes was compared with that of the control group after normalizing to C_t values of GAPDH. Except for the P53 primer sequence, the target gene primer sequences were obtained from OriGene Technologies (Rockville, MD, USA). Bassiony et al. [20] provided the P53 primer sequence. Supplementary Table S1 shows the target gene primer sequences.

Western blotting was performed using whole-cell lysates from Hepa-1c1c7 stable cells (vector and HA-tagged TgIST). Whole-cell proteins were extracted using M-PER^TM mammalian protein extraction reagents (Thermo Fisher Scientific, Waltham, MA, USA). The extracted proteins were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes at 80 V for 15 min and 150 V for 1 h under ice-cold conditions using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). To block unspecific binding of transferred membranes, the membranes were coated with 5% BSA in Tris-buffered saline (TBS-T) (pH 7.6) with 0.1% Tween-20 for 1 h at room temperature (RT). The blocked membranes were incubated with anti-HA tag antibody (Cell Signaling Technology), anti-STAT1 antibody (Santa Cruz Biotechnology), or anti-β-Actin antibody (Santa Cruz Biotechnology) for 16 h at 4°C. The membranes were then washed thrice with TBS-T before incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (m-IgGK BP-HRP; Santa Cruz Biotechnology or Goat anti-Rabbit IgG (H+L)-HRP; Invitrogen) for 1 h at RT. Targets on the membrane were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). The images were captured using Amersham Imager 600 (GE Healthcare, Chicago, IL, USA), and signal intensities were calculated using ImageJ (Free software provided by the National Institutes of Health).

Total proteins were isolated from 2×10⁶ cells of Hepa-1c1c7 stable cells (vector and HA-tagged TgIST) using 400 μl of M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, USA). Next, 100 μl of protein A/G-plus agarose beads (Santa Cruz, Biotechnology) were prereacted with 5 μl of anti-HA tag Ab (Cell Signaling) for 16 h at 4°C. Subsequently, 0.5 mg of total protein extract was added and incubated for 4 h at 4°C. After the supernatants were removed, the anti-HA tag Ab-conjugated protein A/G beads were analyzed through western blotting using anti-STAT1 Ab (Santa Cruz, Biotechnology) and anti-β-Actin Ab (Santa Cruz Biotechnology).

All statistical analyses were performed using GraphPad Prism 5 (GraphPad, La Jolla, CA, USA). The Wilcoxon matched-pairs signed-rank test was used to analyze real-time qPCR data in Hepa-1c1c7 stable cells. A one-way ANOVA followed by a Bonferroni post hoc comparison test was used for the data analysis of real-time qPCR in IFN-γ- or TNF-α-treated Hepa-1c1c7 vector cells. A two-way ANOVA followed by a Bonferroni post hoc comparison test was used to analyze the viability of Hepa-1c1c7 stable cells. Data are expressed as mean±standard deviation (SD). The following significance was assigned: *significant at P<0.05 difference between Hepa-1c1c7 vector cells and Hepa-1c1c7 TgIST cells; ^†significant at P<0.05 difference between nontreated Hepa-1c1c7 vector cells and IFN-γ-, TNF-α-, and Both-treated Hepa-1c1c7 vector cells.

To create the TgIST-expressing retroviral vector, the TgIST (2,673 bp) gene was inserted into the pBABE-HAII plasmid and confirmed using conventional PCR (Fig. 1A). TgIST expression in Hepa-1c1c7 cells was confirmed using PCR and western blotting (Fig. 1B and C). IP assay revealed a sustained binding interaction between TgIST and STAT1 in Hepa-1c1c7 stable cells (Fig. 1D). The effects of TgIST on cell proliferation were investigated using the CCK-8 assay. Intracellular TgIST expression had no significant effect on the viability of Hepa-1c1c7 cells (P<0.05, Fig. 1E). These results showed that Hepa-1c1c7 cells were continuously expressed TgIST. In the basal state of hepatocytes, TgIST does not affect cell viability (Fig. 1E).

We compared the expression of STAT1-mediated inflammatory and apoptotic genes in Hepa-1c1c7 stable cells. In IFN-γ/TNF-α-stimulated factors, the expression of STAT1, interferon regulatory factor 1 (IRF-1), nuclear factor kappa B (NF-κB), and cyclooxygenase 2 (COX2) was significantly reduced in TgIST cells (P<0.05, Fig. 2A). CXC motif chemokine ligands (CXCLs), class II major histocompatibility complex transactivator (CIITA), inducible nitric oxide synthase (iNOS), and suppressor of cytokine signaling 1 (SOCS1) were also studied in response to STAT1 activation. The expression levels of CXCL9/10/11, CTIIA, and iNOS were significantly lower in TgIST cells (P<0.05, Fig. 2B). However, SOCS1, which inhibits the biological effects of proinflammatory cytokines by activating the janus kinase (JAK)/STAT1 pathway, differed insignificantly (P<0.05, Fig. 2B). After analyzing the expression of caspases, P53, B-cell lymphoma-2 (BCL-2), and BCL-2-associated X (BAX) related to hepatocyte apoptosis occurring in sterile inflammatory liver injury, the proapoptotic molecules (Caspase 3/8/9, P53, and BAX) were significantly increased in TgIST cells (P<0.05, Fig. 2C). In contrast, BCL-2, which is crucial in promoting cell survival and inhibiting the action of proapoptotic proteins, was not changed (P<0.05, Fig. 2C). Excessive inflammation damages hepatocytes, increasing hepatic adhesion and leukocyte infiltration. The expression of adhesion markers (Intracellular adhesion molecule 1; ICAM-1 and vascular cell adhesion molecule 1; VCAM-1) and infiltration markers (Programmed death-ligand 1; PD-L1) was significantly lower in TgIST cells (P<0.05, Fig. 2D). These results show that TgIST suppresses the STAT1-mediated proinflammatory and proapoptotic response by binding to and inhibiting STAT1.

The cellular concentration of IFN-γ/TNF-α treatment was evaluated to provide hepatocytes with an enhanced IFN-γ/TNF-α environment for sterile inflammatory liver injury. First, vector cells were treated with different concentrations of IFN-γ/TNF-α (0, 100, 200, 400, 800, and 1,000 ng/ml) before analyzing the expression of STAT1 and IRF-1. Given the changes in gene expression based on concentration, 100 ng/ml was determined as the final concentration to be employed in the following tests (Data not shown). Hepa-1c1c7 stable cells were treated with a previously determined dose of 100 ng/ml IFN/TNF, and TgIST was tested to determine whether it could continue downregulating STAT1-mediated signaling genes in an IFN/TNF-stimulated environment.

In the case of IFN-γ/TNF-α-stimulated factors, the expression of all genes was considerably elevated in IFN-γ/TNF-α-stimulated vector cells (P<0.05, Fig. 3A). In contrast, in TgIST cells, STAT1 and NF-κB did not increase even after IFN-γ/TNF-α stimulation. IRF-1 and COX2 levels increased slightly, but the difference was insignificant (P<0.05, Fig. 3A). In STAT1-mediated inflammatory reactive factors, including CXCLs, CIITA, iNOS, and SOCS1, the expression of all genes was also considerably enhanced in IFN-γ/TNF-α-stimulated vector cells (P<0.05, Fig. 3B). Except for CXCL9 and CXCL10, TgIST cells increased insignificantly in response to IFN-γ/TNF-α stimulation, although the difference was small (P<0.05, Fig. 3B). Although SOCS1 is a negative feedback inhibitor of JAK/STAT cytokine signaling, IFN-γ-mediated STAT1 increases SOCS1 transcription [21].

Because of assessing the expression of apoptosis signaling factors, proapoptotic genes (Caspase 3/8/9, P53, and BAX) were considerably elevated in vector cells (P<0.05, Fig. 3C). However, these genes were not altered (Caspase 3/8), decreased (Caspase 9), or modestly elevated (P53) in TgIST cells (P<0.05, Fig. 3C). In the case of BCL-2, an antiapoptotic gene was dramatically lowered in IFN-γ/TNF-α-treated vector cells but significantly increased in IFN-γ/TNF-α-stimulated TgIST cells (P<0.05, Fig. 3C). Meanwhile, the expression of leukocyte adhesion-related genes (ICAM-1 and VCAM-1) and infiltration-related genes (PD-L1) increased in vector cells but not in TgIST cells (P<0.05, Fig. 3D).

These findings indicate that when hepatocytes are stimulated by IFN-γ/TNF-α, as occurs in sterile inflammatory liver injury, TgIST can continuously suppress the expression of proinflammatory, proapoptotic, and hepatic adhesion/infiltration genes via activated STAT1.

We analyzed whether inhibiting STAT1-mediated signaling mechanisms using TgIST could improve the attenuation of hepatocyte proliferation stimulated by IFN-γ/TNF-α. The CCK-8 assay was used to assess the viability of Hepa-1c1c7 stable cells over time after stimulation with IFN-γ/TNF-α (Fig. 4). In vector cells, cell proliferation was significantly lower than that in the nonstimulated group (Vector+none) starting at 48 h after stimulation with IFN-γ/TNF-α (P<0.05, Fig. 4A). In contrast, in TgIST cells, cell proliferation differed insignificantly from that in the nonstimulated group (TgIST+none) at any time point despite stimulation with IFN-γ/TNF-α (P<0.05, Fig. 4B). Therefore, TgIST improves the attenuated hepatocyte proliferation induced by IFN-γ/TNF-α stimulation and sustains cell growth.